Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S-transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.

Classical swine fever (CSF) caused by classical swine fever virus (CSFV) has resulted in severe losses to the pig industry worldwide. CSFV non-structural protein 4B (NS4B) plays a crucial role in CSFV replication and pathogenicity. However, the function of NS4B is still limited during CSFV infection. In this study, the RNA-seq was used to investigate differentially expressed genes (DEGs) in 3D4/21 cells expressing CSFV NS4B. 4397 DEGs were identified in 3D4/21 cells expressing NS4B compared to cells expressing the empty vector (NC). Twelve DEGs were selected and further verified by RT-qPCR. Enrichment analyses of GO annotations and KEGG pathways revealed that these DEGs were associated with endocytosis, autophagy, cell adhesion, transport, immune response, apoptosis and so on. The expression of endocytosis-related genes, including CAV1/2, CAVIN2, Rab1B, CHMP2B/4C, VPS35, SNX2, Rab11B, CHMP6, MVB12B and VPS28, were found to be regulated. In addition, some genes associated with host immune defense, such as USP15, DHX29, DDX3, RIG-I and MDA5, were downregulated and the genes associated with host autophagy, such as WIPI2, ATG16L2, SMCR8, RPTOR and MLST8, were upregulated. Therefore, CSFV NS4B involved in virus invasion and intracellular trafficking, the induction of autophagy and the inhibition of antiviral response. Taken together, this study provides useful information for further understanding the function of NS4B during CSFV infection.